ABSTRACT. The major thrust of our laboratory is to elucidate the molecular mechanisms underlying regulation of signal transduction pathways by integrins and the cytoskeleton. When normal fibroblasts, epithelial cells, or endothelial cells are deprived of integrin-mediated anchorage to the extracellular matrix (ECM) they fail to progress through the cell cycle. Over the last few years it has become clear that this is based, in large part, on the reduced efficiency of signaling processes in non-anchored cells. We have recently published that signaling in the pathway leading from receptor tyrosine kinases to activation of the Erk/MAP Kinase is regulated by cell anchorage at the level of the Raf-1/Mek/Erk module. We have also shown that protein kinase A (PKA) and p21 activated kinase (PAK) play key roles in this regulation. Thus Aim I of this proposal will determine the molecular basis of anchorage regulation of the Erk pathway by PKA and PAK. Integrin-mediated cell anchorage leads to the formation of complex cytoskeletal structures termed focal contacts. While it is clear that focal contacts play an important role in the regulation of signaling, the contribution of individual focal contact proteins to this process is obscure. In Aim II we will address this issue using emerging new technologies. Thus, we will utilize both RNA interference and antisense oligonucleotides to markedly reduce expression levels of individual focal contact proteins. We will also use new techniques for peptide/protein delivery to selectively block protein-protein interactions within the focal contact. We will then evaluate the impact of these changes on the Erk/MAP Kinase pathway. Most cells are permanently anchored to the ECM via integrins. By contrast, bone marrow-derived cells such as lymphocytes and monocytes normally traffic from a suspended state in the blood to an anchored state in tissues. Thus the relationship between integrin-mediated adhesion and signaling may be different in these cells. We pursue this issue in Aim III. In preliminary experiments we have found that integrin-mediated cell adhesion regulates chemokine signaling to the Erk pathway in lymphoid cells. However, the locus of regulation is quite different from that seen in fibroblasts. In this Aim we will pursue the mechanism underlying integrin regulation of chemokine signaling, and will also explore its biological ramifications.